Periodic Reporting for period 2 - TITINmap (Towards Uncovering the Splicing Code of the Gigantic Gene Titin in Familial Dilated Cardiomyopathy)
Reporting period: 2023-01-01 to 2023-12-31
Titin (TTN) is the largest protein of the human body and critical for the contraction of heart muscle cells. TTN mutations are prevalent in patients with familial dilated cardiomyopathy (DCM), a common heart disease that is a global threat to the aging society. RBM20, which is also mutated in patients with an aggressive form of DCM, regulates the length and function of TTN by a process named alternative splicing. How RBM20 mediates alternative splicing of TTN is largely unknown. My fellowship project was to develop new methods to analyze RBM20-dependent splicing of TTN and other crucial targets in single cells. To this end, I performed CRISPR-based gene editing in human and mouse cardiomyocytes to repair Rbm20 mutations and analyze the outcome on TTN splicing. I showed that the splicing of TTN and other RBM20 target genes was successfully restored and consequently the DCM-phenotype was alleviated in juvenile mice. Secondly, I sought to analyze changes in the repertoire of TTN isoforms upon disease-relevant mutations in the RBM20 gene. To this end, I harnessed the power of long-read sequencing and split-pool barcoding to establish a single-cell sequencing method whereby I could measure changes in alternative splicing in single cells. Using this method, I analyzed Rbm20-dependent splicing in human cardiomyocytes which can be used to construct a comprehensive splice map of TTN and other isoforms that are expressed in patients with Rbm20 mutations. I envision that knowledge of such a splice map can be exploited for developing therapeutic strategies to revert aberrant alternative splicing in patients with DCM.
The main results of my fellowship project are:
1. Generation and validation of a TTN splice reporter induced pluripotent stem cells (iPSCs) using EXCISER.
Exploitation and dissemination: I have share the cell line with my colleagues in the Steinmetz laboratory who are aiming to use this model as basis for high-throughput saturation screens proposed as part of another grant funded by the European Commission (CARDIOREPAIR). To this end, I also shared this cell line with our collaborator Prof. Gil Westmeyer who is named in the grant to perform initial proof-of-concept experiments.
2. Establishing in vivo base pair correction of Rbm20 in the adult murine heart with read-out of TTN splicing (amongst other physiological DCM assays).
Exploitation and dissemination: I summarized this part of the project in a manuscript which was published at Biorxiv (https://doi.org/10.1101/2022.12.13.520227(opens in new window)) and has now been accepted at Nature Communications. I presented this project in several group talks, retreats and most recently in the gene therapy department at Roche. Based on these results, we have several ongoing projects in the Steinmetz group to perform gene editing in pigs and to dissect the efficacy of gene editing in vivo.
3. Establishing a method for analysing alternative splicing in single cardiomyocytes.
Exploitation and dissemination: The protocol for this method has been shared with the Steinmetz group and external collaborators including Victoria Parikh (Stanford University) and Wu Wei (Lingang Laboratory, Shanghai). They will use this method for high-throughput interrogation of isoform changes upon mutations in Rbm20.
1. Generation and validation of a TTN splice reporter induced pluripotent stem cells (iPSCs) using EXCISER.
Exploitation and dissemination: I have share the cell line with my colleagues in the Steinmetz laboratory who are aiming to use this model as basis for high-throughput saturation screens proposed as part of another grant funded by the European Commission (CARDIOREPAIR). To this end, I also shared this cell line with our collaborator Prof. Gil Westmeyer who is named in the grant to perform initial proof-of-concept experiments.
2. Establishing in vivo base pair correction of Rbm20 in the adult murine heart with read-out of TTN splicing (amongst other physiological DCM assays).
Exploitation and dissemination: I summarized this part of the project in a manuscript which was published at Biorxiv (https://doi.org/10.1101/2022.12.13.520227(opens in new window)) and has now been accepted at Nature Communications. I presented this project in several group talks, retreats and most recently in the gene therapy department at Roche. Based on these results, we have several ongoing projects in the Steinmetz group to perform gene editing in pigs and to dissect the efficacy of gene editing in vivo.
3. Establishing a method for analysing alternative splicing in single cardiomyocytes.
Exploitation and dissemination: The protocol for this method has been shared with the Steinmetz group and external collaborators including Victoria Parikh (Stanford University) and Wu Wei (Lingang Laboratory, Shanghai). They will use this method for high-throughput interrogation of isoform changes upon mutations in Rbm20.
The impact of this research project is manifold. First, I have generated the first endogenous splice reporter of TTN which can be used to dissect alternative isoforms of TTN in unprecedented detail and at single cell resolution. Notably, we seek to perform high-throughput CRISPR interference screens to identify other genes that regulate the splicing of TTN. Other plans are to use this cell line as read-out for saturation mutagenesis screens to uncover mutations of RBM20 that affect splicing. This is subsequently very important to stratify novel disease-causing mutations in patients with DCM. Finally, the TTN splice reporter cell line can be used in combination with small molecule screening platforms to identify drugs that could revert the aberrant splice profile of TTN concomitant with mutations in RBM20. Secondly, in our paper, we developed an approach for correcting Rbm20 mutations directly in the heart of adult mice. We identified a cohort of RBM20-responsive genes which can be used as biomarkers for screening the magnitude of different RBM20 mutations. This project has broad impact on general gene therapy strategies for hereditary cardiac mutations. Finally, a novel method based on combinatorial barcoding and long-read sequencing was established. This method is broadly applicable to analyze alternative splicing in single cells. In contrast to several other methods, there is no cell size limitation which enables the isoform analysis in large cells such as cardiomyocytes, neurons, and others. In summary, this project pushed the technological limits of several aspects in the fields of transcriptomics, cell engineering and gene therapy and therefore has a broad impact in these areas.